Method for detecting leptin receptor ligands

ABSTRACT

The present invention relates to a method for detecting leptin receptor ligands by measuring the energy transfer between fusion proteins comprising a leptin receptor and an energy donor protein or an energy acceptor acceptor protein proteins. The present invention also relates to fusion proteins for implementing said method.

The present invention relates to a method for detecting leptin receptorligands using the energy transfer between fusion proteins composed ofleptin receptors and of energy-donor or energy-acceptor proteins.

It also relates to fusion proteins for implementing this method.

Leptin is a protein having a molecular weight of 16 kDa which issecreted by adipocytes. This protein is associated with the feeling ofsatiety, and plays a major role in the control of body weight, energyconsumption, bone formation and angiogenesis, but also in otherphysiological functions, such as the triggering of puberty and thecontrol of reproduction, or the regulation of the T-lymphocyte-mediatedimmune response.

The leptin receptor (OBR) belongs to the family of cytokine receptors.It is composed, as illustrated in FIG. 1, of a single polypeptide chaincomprising a transmembrane domain (Tartaglia et al., J. Biol. Chem.,272, 6093-6096, 1995). Patent application WO 97/19952 relates to thisreceptor.

Six different isoforms of the OBR, having C-terminal domains withdifferent lengths, have been described. These isoforms all derive from asingle gene, by alternative splicing. There also exists a soluble formof OBR containing the leptin-binding site, which corresponds to theextracellular domain of the membrane-bound form. This soluble form,generated post-translationally by proteolysis at the plasma membranefrom the membrane-bound forms, is found in the blood.

Another soluble form of the OBR, resulting from a mutation generating astop codon before the transmembrane domain, is also found is certainvery rare cases.

A fusion protein consisting of the long form of the leptin receptor(OBR1) fused to EGFP (Enhanced Green Fluorescence Protein) has been usedby Lundin et al. (Biochemica and Biophysica Acta, 1499, 130-138, 2000)to study the location of the receptor.

Activation of the OBR is thought to take place via a tetrameric complexcomposed of two janus kinases 2 (JAK2) and two OBR. Leptin-inducedactivation of the receptor will induce a change in the conformation ofthe OBR, which would itself activate a JAK2, which in turn wouldtrans-phosphorylate another JAK2 and then the OBR receptor.

Activation of the OBR appears to be responsible for all the knowneffects of leptin, such as weight loss, and all the phenomena involvedin weight disorders.

The inhibitory properties of leptin with respect to bone synthesis havethus recently been demonstrated. Leptin acts by inhibiting the activityof osteoblasts, a population of cells responsible for the formation ofbones.

Modifying leptinemia might make it possible to treat diseases associatedwith a decrease in bone density, such as, for example, osteoporosis, or,conversely, those associated with considerable calcification.

In 1999, Xu et al. (Proc. Natl. Acad. Sci. USA 96, 151-156) described amethod for detecting protein-protein interactions in living cells. Thismethod is also the subject of patent application WO 99/66324.

This method, called BRET (for Bioluminescent Resonance Energy Transfer)is based on a natural phenomenon, the emission of fluorescence by marineorganisms. The enzymatic transformation, by Renilla luciferase (Luc), ofa substrate which can cross the membrane generates a bioluminescencewhich, in turn, excites an energy acceptor such as yellow fluorescentprotein (YFP). This method corresponds to the LRET (for LuminescentResonance Energy Transfer) described by Wang et al. (Mol. Gen. Genet.264: 578-587 (2001)).

The efficiency of the energy transfer depends on the physical proximityand on the respective orientations of the acceptor and of the donor.Thus, the coexpression of luciferase and of YFP is not sufficient toinduce an energy transfer since the distance between the two partnersmust be less than 100 Å. In order to study the interaction between twopotential interaction partners, the first protein was fused toluciferase and the second protein to YFP. If the two proteins interact,an energy transfer can be observed.

Since then, the BRET method has been used on a limited number ofreceptors, having a structure very different from the leptin receptor.Thus, some authors describe the use of the method on receptors of the Gprotein coupled receptor (GPCR) family, such as the β 2-adrenergic(Angers et al., 2000, Proc. Natl. Acad. Sci. USA 10, 1073),cholesystocine type A (CCK; Cheng et al., 2001, Biol. Chem. 276:48040-48047), and thyrotropin-releasing hormone (Kroeger et al., 2001,J. Biol. Chem. 276: 12736-12743) receptors.

These receptors, which are large in size, exhibit a complex structurecomprising 7 transmembrane domains. Finally, Boute et al. (2001, Mol.Pharmacol. 60: 640-645) have described the following of activation ofthe insulin receptor using BRET.

The insulin receptor consists of covalent dimers, and not of noncovalentcomplexes like the leptin receptor. In addition, it comprises quite along intracellular portion. Finally, the authors show that the change inBRET induced by insulin can only be implemented on the solubilizedreceptor.

In a few decades, obesity has become a major public health problem inindustrialized countries, where it now affects 20 to 30% of thepopulation. These numbers should further increase alarmingly in theyears to come. Due to its multifactorial causes, which originate togreater or lesser degrees among, firstly, environmental factors (dietarybehavior, access to food, energy expenditure, etc.) and, secondly,multiple genetic causes, obesity constitutes a real challenge formedicine.

Similarly, bone diseases, such as osteoporosis, affect an increasinglylarge portion of the population. The discovery of novel molecules fortreating the various diseases associated with the leptin receptor, suchas obesity and osteoporosis, therefore represents high stakes for publichealth.

However, no method for specifically screening leptin receptor agonistsor antagonists exist, which can be used at high throughput.

The applicants have therefore endeavored to implement a rapid, specificand effective screening test for leptin receptor agonists orantagonists.

They have shown, surprisingly, that the change in BRET induced by leptincan be used on one of the isoforms of the leptin receptor, but that itcannot be implemented with all the isoforms.

They have also shown that the implementation of BRET on the leptinreceptor is optimal under certain operating conditions.

The present invention therefore relates to a method for detecting leptinreceptor ligands using the resonance energy transfer between a firstfusion protein composed of a leptin receptor, or of a substantialportion of a leptin receptor, and of an energy donor protein, or of asubstantial and active portion of an energy donor protein, and a secondfusion protein composed of a leptin receptor, or of a substantialportion of a leptin receptor, and of an energy acceptor protein, or of asubstantial and active portion of an energy acceptor protein.

It also relates to fusion proteins for carrying out this method, andalso to nucleic acids encoding these proteins.

A subject of the invention is also a method for the curative orpreventive treatment of diseases associated with leptin, consisting inadministering a ligand selected using the method defined above to apatient suffering from said disease.

A first subject of the present invention is therefore a fusion protein,which is composed of a leptin receptor, or of a substantial portion of aleptin receptor, and of an energy acceptor or donor protein, or of asubstantial and active portion of an energy acceptor or donor protein.

The fusion proteins according to the present invention are composed, insubstance, of a portion corresponding to all or part of the sequence ofa leptin receptor and of a portion corresponding to an energy donor oracceptor protein. They may, however, comprise other amino acidsequences, derived from other proteins, such as signal sequences. Thusthe sequence SEQ ID No. 4 consists of a portion of the sequence SEQ IDNo. 2 and of other sequences, and in particular of the signal sequenceof mouse interleukin 3.

Advantageously, the energy donor protein is Renilla luciferase. It may,however, be any other energy donor protein, such that the emissionspectrum of the donor overlaps sufficiently with the excitation spectrumof the acceptor so as to allow efficient energy transfer between the twoparts. It may thus be GFP, if the energy transfer is FRET, or elseaequorin if the energy transfer is CRET. Aequorin can be obtained andused as described in patent application EP 0 187 519, or in the articleby Inouye et al. (PNAS USA 82: 3154-3158 (1985)).

As regards the energy acceptor fluorescent protein, it is preferentiallyDsRed, or GFP or a mutant of this protein, such as YFP, EYFP, wild-typeGFP, GFPS65T, or Topaz.

It may, however, be any other energy acceptor fluorescent protein, suchthat the excitation spectrum of the acceptor and the emission spectrumof the donor overlaps sufficiently to allow efficient energy transferbetween the two partners.

These proteins are known to those skilled in the art, who can find theirsequences in the literature, in particular in the review by Blinks etal. (Pharmacol. Rev. 28: 1-93 (1976)). In particular, GFP is describedby Tsien (Annu. Rev. Biochem. 67: 509-544 (1998)) and the cloningthereof is described by Prasher et al. (Gene 111: 229-233 (1992)). Asregards the cloning of DsRed, it is described by Matz et al. (Nat.Biotechnol. 17:969-973 (1999)). For Rluc, those skilled in the art canrefer to Blinks et al. (Pharmacol. Rev. 28: 1-93 (1976)) or else toLorenz et al. (PNAS 88: 4438-4442 (1991)).

Advantageously, the isoform of the leptin receptor which is entirely orpartly included in the fusion protein is a short isoform, or an isoformexhibiting a short intracellular domain.

Such an isoform advantageously comprises a Box1 intracellular domain,but does not comprise a Box 3 intracellular domain.

Preferentially, this isoform is the OBRs isoform, and even morepreferentially the human OBRs isoform. This isoform may, however, comefrom any other species.

It may also be any other isoform, preferentially short, and even morepreferentially comprising at least the extracellular domain of the OBR,such as the soluble form of the OBR containing the leptin-binding site,described by Lee et al. (Nature 379, 632-635 (1996)), Gavrilova et al.(JBC 272: 30546-30551 (1997)), Maamr. et al. (Endocrinology 142:4389-4393 (2001)) or Clement et al. (Nature 392: 398-401 (1998)).

According to a particularly preferential embodiment, the isoform is thehuman OBRs isoform of sequence SEQ ID No. 2. It may also be a variant ofthis protein, exhibiting at least 65%, preferentially at least 75%, andeven more preferentially at least 85% or 95%, identity with the sequenceSEQ ID No. 2.

The fusion protein may comprise only a portion of the human OBRsisoform. Advantageously it comprises the portion between amino acids 46and 866 of the sequence SEQ ID No. 2.

The portion corresponding to the leptin receptor may thus have thesequence SEQ ID No. 4, or a variant of this sequence exhibiting at least65%, preferentially at least 75%, and even more preferentially at least85% or 95%, identity.

Particularly advantageously, the donor fusion protein has the sequenceSEQ ID No. 6, or a variant of this sequence exhibiting at least 65%identity.

Particularly advantageously, the acceptor fusion protein has thesequence SEQ ID No. 8, or a variant of this sequence exhibiting at least65% identity.

Other subjects of the present invention are nucleic acids encoding theseproteins. Such nucleic acids may be complementary or genomic DNAs, orRNAS. These nucleic acids or polynucleotides may be in single-strandedform or in the form of a duplex.

They are particularly advantageously complementary DNAs.

Preferentially, a subject of the invention is a nucleic acid having atleast 65%, preferentially at least 75%, and even more preferentially atleast 85% or 95%, nucleotide identity with a nucleic acid of sequenceSEQ ID No. 5 or SEQ ID No. 7.

According to yet another aspect, the invention relates to a nucleic acidwhich hybridizes, under high stringency hybridization conditions, with anucleic acid as defined above, and more particularly a nucleic acid ofnucleotide sequences SEQ ID No. 5 and SEQ ID No. 7, or a nucleic acid ofcomplementary sequence.

For the purpose of the present invention, the “percentage identity”between two nucleotide or amino acid sequences can be determined bycomparing two optimally aligned sequences through a window ofcomparison.

The portion of the nucleotide or polypeptide sequence in the window ofcomparison can thus comprise additions or deletions (for example gaps)relative to the reference sequence (which does not comprise theseadditions or these deletions) so as to obtain optimal alignment of thetwo sequences.

The percentage is calculated by determining the number of positions atwhich an identical nucleic acid base or amino acid residue is observedfor the two (nucleic acid or peptide) sequences compared, then dividingthe number of positions at which there is identity between the two basesor amino acid residues by the total number of positions in the window ofcomparison, then multiplying the result by 100 in order to obtain thepercentage sequence identity.

The optimal alignment of the sequences for the comparison can be carriedout on a computer using known algorithms contained in the WISCONSINGENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 ScienceDrive, Madison, Wis.

By way of illustration, the percentage sequence identity may be effectedusing the BLAST program (versions BLAST 1.4.9 of March 1996, BLAST 2.0.4of February 1998 and BLAST 2.0.6 of September 1998), using exclusivelythe default parameters (S. F. Altschul et al., J. Mol. Biol. 1990 215:403-410, S. F. Altschul et al., Nucleic Acids Res. 1997 25: 3389-3402).Blast searches for sequences similar/homologous to a reference “request”sequence, using the algorithm of Altschul et al. The request sequenceand the databases used may be peptide- or nucleic acid-related, anycombination being possible.

For the purpose of the present invention, the expression “highstringency hybridization conditions” will be intended to mean thefollowing conditions:

1-Membrane Competition and Prehybridization:

-   -   40 μl of salmon sperm DNA (10 mg/ml)+40 μl of human placental        DNA (10 mg/ml) are mixed.    -   The mixture is denatured for 5 min at 96° C., and then immersed        in ice.    -   The 2×SSC is removed and 4 ml of formamide mix are poured into        the hybridization tube containing the membranes.    -   The mixture of the two denatured DNAs is added.    -   Incubation is carried out at 42° C. for 5 to 6 hours, with        rotation.        2-Labeled Probe Competition:    -   10 to 50 μl of Cot I DNA, depending on the amount of        repetitions, are added to the labeled and purified probe.    -   Denaturation is carried out for 7 to 10 min at 95° C.    -   Incubation is carried out at 65° C. for 2 to 5 hours.        3-Hybridization:    -   The prehybridization mix is removed.    -   40 μl of salmon sperm DNA+40 μl of human placental DNA are        mixed; the mixture is denatured for 5 min at 96° C., and then        immersed in ice.    -   4 ml of formamide mix, the mixture of the two DNAs and the        denatured labeled probe/Cot I DNA are added to the hybridization        tube.    -   Incubation is carried out for 15 to 20 hours at 42° C., with        rotation.        4-Washes:    -   One wash is carried out at ambient temperature in 2×SSC, to        rinse.    -   Two 5-minute washes are carried out at ambient temperature,        2×SSC and 0.1% SDS at 65° C.    -   Two 15-minute washes are carried out at 65° C., 1×SSC and 0.1%        SDS at 65° C.        The membranes are wrapped in Saran wrap and exposed.

The hybridization conditions described above are suitable for thehybridization under high stringency conditions of a nucleic acidmolecule of variable length of 20 nucleotides for several hundrednucleotides.

It goes without saying that the hybridization conditions described abovecan be adjusted as a function of the length of the nucleic acid thehybridization of which is sought, or of the type of labeling chosen,according to techniques known to those skilled in the art.

The suitable hybridization conditions may, for example, be adjustedaccording to the teaching contained in the work by HAMES and HIGGINS(1985, “Nucleic acid hybridization: a practical approach”, Hames andHiggins Ed., IRL Press, Oxford) or else in the work by F. AUSUBEL et al.(1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y.).

The proteins which are the subject of the present invention can beobtained by any means known to those skilled in the art. They are,however, advantageously obtained by expression of the nucleic acids asdescribed above, encoding these proteins, optionally inserted intoexpression vectors, in advantageously chosen cells, optionally followedby an extraction or a purification which may be total or partial.

The invention also relates to a recombinant vector comprising a nucleicacid according to the invention.

Advantageously, such a recombinant vector will comprise a nucleic acidchosen from the following nucleic acids:

a) a nucleic acid encoding a protein having at least 65% amino acididentity with a sequence SEQ ID No. 6 or SEQ ID No. 8, or a peptidefragment or a variant thereof;

b) a nucleic acid comprising a polynucleotide having a sequence SEQ IDNo. 5 or SEQ ID No. 7, or a fragment or a variant thereof;

c) a nucleic acid having at least 65% nucleotide identity with a nucleicacid having a sequence SEQ ID No. 5 or SEQ ID No. 7, or a fragment or avariant thereof;

d) a nucleic acid which hybridizes, under high stringency hybridizationconditions, with a nucleic acid of sequence SEQ ID No. 5 or SEQ ID No.7, or a fragment or a variant thereof.

For the purpose of the present invention, the term “vector” will beintended to mean a circular or linear DNA or RNA molecule which isindifferently in single-stranded or double-stranded form.

According to one embodiment, the expression vector comprises, besides anucleic acid in accordance with the invention, regulatory sequences fordirecting the transcription and/or the translation thereof.

According to an advantageous embodiment, a recombinant vector accordingto the invention will in particular comprise the following elements:

(1) elements for regulating the expression of the nucleic acid to beinserted, such as promoters and enhancers;

(2) the coding sequence included in the nucleic acid in accordance withthe invention to be inserted into such a vector, said coding sequencebeing placed in phase with the regulatory signals described in (1); and

(3) suitable transcription initiation and stop sequences.

In addition, the recombinant vectors according to the invention mayinclude one or more origins of replication in the cellular hosts inwhich their amplification or their expression is desired, markers orselectable markers.

By way of example, the promoters for eukaryotic cells will comprise theHSV virus thymidine kinase promoter or else the mouse metallothionein-Lpromoter.

In general, for the choice of a suitable promoter, those skilled in theart may advantageously refer to the work by SAMBROOK et al. (1989,“Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) or else to the techniquesdescribed by FULLER et al. (1996, Immunology in Current Protocols inMolecular Biology, Ausubel et al.).

The preferred vectors according to the invention are plasmids, such as,for example, vectors pCDNA3 (Invitrogen), pQE70, pQE60, pQE9 (Qiagen),psiX174, pBluescript SA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT,pOG44, pXTI or pSG (Stratagene).

They may also be vectors of the baculovirus type, such as the vectorpVL1392/1392 (Pharmingen) used to transfect cells of the Sf9 line (ATCCNo. CRL 1711) derived from Spodoptera frugiperda.

They may also be adenoviral vectors, such as human adenovirus type 2 or5.

A recombinant vector according to the invention may also be a retroviralvector or alternatively an adeno-associated vector (AAV). Suchadeno-associated vectors are, for example, described by FLOTTE et al.(1992, Am. J. Respir. Cell Mol. Biol., 7: 349-356).

Subjects of the present invention are also cells comprising a protein, anucleic acid or a vector as described above, or fragments of thesecells, lysates of these cells or else membranes of these cells.

Such cells may be cells isolated from an organism and cultured in anappropriate growth medium. They are, however, preferentially cell lines.Thus, such lines are particularly advantageously the cell lines HEK 293,COS (ATCC No. CRL 1650), COS-M6 and HeLa (ATCC No. CCL2), or else Cv 1(ATCC No. CC170), Sf-9 (ATCC No. CRL 1711), CHO (ATCC No. CCL-61) or 3T3(ATCC No. CRL-6361).

The membranes of these cells can be prepared by any method known tothose skilled in the art. Preferentially, they will be prepared bymechanical grinding of the cells, and then centrifugation of thesuspensions obtained, as illustrated in the examples which follow.

The present invention also relates to compositions comprising cells asdescribed above and saponin.

The present invention also relates to a method for determining thebinding of compounds to the leptin receptor, comprising the stepsconsisting in:

-   -   bringing said compound into contact with an energy donor fusion        protein as described above and an energy acceptor fusion protein        as described above, or cells, or fragments or lysates or        membranes of cells comprising such a protein, and an appropriate        enzyme substrate, and    -   measuring the energy transfer.

Preferentially, said method is used with cells treated with saponin.

The energy donor fusion proteins and the energy acceptor fusion proteinsare chosen such that the energy resulting from the activation of thedonor may be transferred efficiently to the acceptor

In an advantageous embodiment of said method, the energy donor fusionprotein is a protein from fusion between the leptin receptor, or asubstantial portion of the leptin receptor, and luciferase, or asubstantial portion of luciferase, in which case the substrate isadvantageously coelenterazine.

In a preferential embodiment of said method, the energy acceptor fusionprotein is a protein from fusion between the leptin receptor, or asubstantial portion of the leptin receptor, and YFP, or a substantialportion of YFP.

In an advantageous embodiment of said method, the energy transfermeasured in the presence of the test compound is compared to thatmeasured in the absence of the test compound.

Preferentially, the method is used on cell membranes as described above.

Preferentially, the donor and acceptor proteins according to the presentinvention are chosen such that the energy transfer takes place by BRETor LRET resonance. However, such an energy transfer may be effected byFRET (Fluorescent Resonance Energy Transfer) or else by CRET(Chemiluminescent Resonance Energy Transfer).

Whatever the type of energy transfer, the energy donor fusionprotein/energy acceptor fusion protein pairs are chosen so as to allowsuch a transfer.

CRET consists of energy transfer between aequorin, which is aluciferase, and GFP.

FRET consists of energy transfer between two proteins of the GFPfamilies having different spectra. For the implementation of thesetransfers, those skilled in the art may refer to Baubet et al. (PNAS USA97: 7260-7265 (2000)) for CRET, to Matyus (J. Photochem. Photobiol. B12: 323-337 (1992)) and Pollok and Heim (Trends Cell Biol. 9: 57-60(1999)) for FRET.

Another subject of the present invention is a method for screening ordetecting compounds intended for the prevention and/or treatment ofpathological conditions associated with leptin, comprising the stepsconsisting in:

-   -   bringing said compound into contact with an energy donor fusion        protein as described above and an energy acceptor fusion protein        as described above, or cells in the absence or presence of        saponin, or fragments or lysates or membranes of cells,        comprising such proteins, and optionally an appropriate enzyme        substrate, and    -   measuring the energy transfer.

Such a method may be used for screening leptin receptor agonists orantagonists.

The method according to the present invention is compatible with the96-well or 384-well plates generally used. It does not require the useof radio-active molecules, is sensitive, reproducible and rapid, and theresult is easy to read. Specifically, this method has a goodsignal/background noise ratio and low cross reactivity with ligandsother than leptin. This is explained at least partially by the fact thatthe activity of the OBR is detected directly at the level of thereceptor, which makes it possible to eliminate possible sources of crossreactivity at other levels of the signaling pathways, as can be observedin the case of assays based on reporter genes or on cell growth. Inaddition, this method is not limited to a transduction pathway having aspecific signal, but, on the contrary, is capable of detecting allmolecules which interact with the OBR.

This characteristic is particularly advantageous for carrying outlarge-scale screening, since an increasing number of membrane receptorligands are found to activate some pathways but not other pathways.

The present invention also relates to the use of compounds selectedusing a method consisting in:

-   -   bringing said compound into contact with an energy donor fusion        protein and an energy acceptor fusion protein as described        above, or cells, or fragments or lysates or membranes of cells        comprising such a protein, and optionally an appropriate enzyme        substrate, and    -   measuring the energy transfer, for producing a medicinal product        for the curative or preventive treatment of diseases associated        with leptin or with its receptor.

Finally, a subject of the invention is a method for the curative orpreventive treatment of diseases associated with leptin or with itsreceptor, comprising the steps of:

-   -   selecting said compound using a method consisting in:    -   bringing said compound into contact with an energy donor fusion        protein and an energy acceptor fusion protein, or cells, or        fragments, lysates or membranes of cells comprising such a        protein, and an appropriate enzyme substrate, and    -   measuring the energy transfer, and    -   of administering said compound to a patient suffering from said        disease.

Such diseases may be diseases associated with a decrease in bonedensity, such as, for example, osteoporosis, or, conversely, thoseassociated with considerable calcification.

They may also be diseases having an effect on weight, such as obesity,diabetes or anorexia. The compounds of the invention may be formulatedin pharmaceutical compositions for the purpose of topical, oral,parenteral, intranasal, intravenous, intra-muscular, subcutaneous,intraocular, etc. administration. Preferentially, the pharmaceuticalcompositions contain pharmaceutically acceptable vehicles for aninjectable formulation. They may in particular be salines (monosodiumphosphate, disodium phosphate, sodium chloride, potassium chloride,calcium chloride or magnesium chloride, etc., or mixtures of suchsalts), sterile, isotonic solutions, or dry, in particular lyophilized,compositions which, by addition, as appropriate, of sterilized water orof physiological saline, make it possible to constitute injectablesolutes.

Finally, the method according to the present invention also makes itpossible to screen serum from obese patients, for the presence orabsence of nonfunctional leptin, or else to screen molecules whichinterfere with the dimerization of the OBR.

FIG. 1 diagrammatically represents the fusion proteins. Box1 representsthe JAK2-binding site; Box3 represents the STAT protein-binding site; TMrepresents the transmembrane domain.

FIGS. 2 a and 2 b illustrate the expression of the OBR constructs in COScells, estimated by radio-labeling experiments using ¹²⁵I-leptin asradio ligand.

In FIGS. 2 a and 2 b, the total cell content of OBR and the percentageof cell surface binding sites are respectively measured.

FIG. 2 c illustrates the cellular location of the expression of theOBR1-YFP and OBRs-YFP construct in the presence and absence of leptin.

FIG. 2 d illustrates the activation of JAK2 with various OBR constructs.FIG. 2 e illustrates the effect of the stimulation with leptin of cellscoexpressing the reporter gene for STAT3 and various OBR constructs.

FIG. 3 illustrates the constitutive dimerization of OBR. HEK 293 cellsexpressing the various OBR constructs indicated are incubated in thepresence of coelenterazine. The energy transfer is measured using aluminometer.

FIGS. 4 a and 4 b illustrate the effect of leptin binding on theconstitutive BRET of the OBR.

FIG. 4 a: HeLa cells expressing the various OBR constructs indicated areincubated in the presence of leptin before initiating the luciferasereaction. The energy transfer is measured using a luminometer.

FIG. 4 b: The effect of leptin is compared in whole cells coexpressingOBRs-Luc and OBR-YFP, in the presence or absence of saponin, in totallysates and in membrane preparations.

FIGS. 5 a to 5 e illustrate the optimization and the characterization ofthe change in BRET induced by leptin on the OBRs. Membranes preparedfrom HeLa or COS cells coexpressing OBRs-Luc and OBRs-YFP werepre-incubated with or without leptin before initiating the luciferasereaction.

FIG. 5 a: Optimization of the relative and absolute levels of expressionof OBRs-Luc and of OBRs-YFP.

FIG. 5 b: Variation of the BRET signal induced with leptin as a functionof time.

FIG. 5 c: BRET/leptin concentration dose-response curves on membrane andintact cells in the presence of saponin (0.05%).

FIG. 5 d: ¹²⁵I-leptin binding competition by augmentation of increasingconcentrations of leptin.

FIG. 5 e: Specificity of the changes in BRET induced with leptin. Themembranes were preincubated with saturating concentrations oferythropoietin (EPO, 10 U/ml), of trombopoietin (TPO, 10 nM), ofgranulocyte macrophage colony stimulating factor (GM-CSF, 250 ng/ml), ofinterleukin 3 (IL3, 280 ng/ml), of interleukin 6 (IL6, 100 ng/ml), ofprolactin (PRL, 200 ng/ml), of stem cell factor α (SCFA, 250 ng/ml), ofepidermal growth factor (EGF, 100 ng/ml), of insulin (Ins, 100 nM), oflipopolysaccharide (LPS, 100 ng/ml) and of tumor necrosis factor α(TNFα, 50 ng/ml).

FIG. 6: Cotransfection of COS cells with a constant amount of OB-Rs-Luc(50 ng) and an increasing amount of OB-Rs-YFP: ∘, 200 ng; ●, 400 ng; Δ,800; ♦, 1600; ⋄, 3 200. The BRET measurements were made on the cells inthe presence of saponin (0.015%), incubated or not incubated withincreasing doses of leptin, and are expressed as mBRET.

The present invention is illustrated, without however being limited, bythe following examples.

Materials and Methods Used In The Examples Plasmid Constructs,Transfections and Cell Culture

The OB-R-YFP and OB-R-Luc fusion proteins were constructed by ligationof YFP and of Luc to the C-terminal end of the receptors by conventionalmolecular biology techniques. The coding regions of YFP obtained fromthe vector pGFPtpz-N1 Cytogem®-Topaze (Packard, Meriden, Conn.) wereinserted into the EcoRV site of pcDNA3/CMV (Invitrogen, Groningen, TheNetherlands), which contains a modified polylinker site. The codingregion of Renilla luciferase was obtained from pRL-CMV (Promega,Madison, Wis.) and inserted into the EcoRV site of pcDNA3 modified. Thecoding regions of OBR1 and OBRs (a gift from Dr. Gainsford, RoyalMelbourne Hospital, Victoria, Australia) were inserted into the twovectors described above, respectively in the EcoR1/BamH1 and Nhe1cloning sites. The stop codons were deleted by site-directed mutagenesisand the frame of the fusion protein was adjusted.

The HEK 293, COS-M6 and HeLa cell lines were cultured in DMEMsupplemented with the following components: 10% (v/v) FBS, 4.5 g/literglucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 mM glutamine(Life Technologies, Gaithersburg, Md.).

The transient transfections were carried out using the FuGene 6transfection reagent (Roche, Basle, Switzerland).

Fluorescence Microscopy

Two days after transfection with the YFP constructs, the cells wereincubated with 100 nM leptin for 60 min and 0.01 mM bisbenzamidine for15 min before being washed in PBS and fixed for 20 min at ambienttemperature in a cold solution of 4% paraformaldehyde in PBS. Thesections were observed by fluorescent microscopy using FITC and DAPIfilters.

Preparation of Membranes and Solubilization

The cells were placed in ice, washed twice in PBS at the temperature ofthe ice and detached mechanically in a buffer 1 (5 mM Tris, 2 mM EDTA,pH 7.4, 5 mg/liter of soybean trypsin inhibitor, 5 mg/liter of leupeptinand 10 mg/liter of benzamidine) at the temperature of ice. The cellsuspensions are homogenized with a Polytron homogenizer (Janke & KunkelUltra-Turrax T25) three times for 5 sec. The lysate is centrifuged at450×g for 5 min at 4° C. and the supernatant is centrifuged at 48 000×gfor 30 min at 4° C. The final pellet is washed twice in buffer 1 andresuspended in a solution (75 mM Tris (pH 7.4), 12.5 mM MgCl₂, 5 mM EDTAwith protease inhibitors, as described above) and immediately used inradioactive ligand-binding experiments or BRET experiments.

Immunoprecipitation of JAK2

HeLa cells were cotransfected with plasmids expressing JAK2 labeled withHA2 (a gift from Dr. Wojchowski, Pennsylvania State University,Pennsylvania, USA) and plasmids containing various OBR constructs. Thecells were lysed in lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 5%glycerol, 0.02% NaN₃, 0.1% NP40, 1 mM orthovanadate, 5 mg/liter ofsoybean trypsin inhibitor and 10 mg/liter of benzamidine) andcentrifuged for 15 min at 13 000 rpm. The soluble fraction isimmuno-precipitated for 2 h with an anti-JAK2 polyclonal antibody(HR-758) (1 μg/ml) (Santa-Cruz Biotechnology, Santa Cruz, Calif.).

SDS-Page Immunoabsorption

The JAK2 immunoprecipitates were denatured in the solution (62.5 mMTris/HCl (pH 6.8), 5% SDS, 10% glycerol and 0.05% bromophenol blue), at100° C. for 10 minutes. The proteins were separated by SDS-PAGE in 7%polyacrylamide, and transferred onto nitrocellulose. The immunodetectionwas carried out with an anti-phosphotyrosine antibody 4G10 (2 μg/ml)(Upstate Biotechnology, Lake Placid, N.Y.). The immunoreactivity wasrevealed using an appropriate secondary antibody coupled to horseradishperoxidase, and the ECL chemiluminescence agent (Amersham, Aylesbury,UK).

¹²⁵I-leptin-binding Assay

Transfected cells were serum depleted in DMEM (1% BSA) 24 h before thebinding experiments. To measure the leptin binding to the surface of thecells, the cells were washed twice with PBS at the temperature of iceand incubated in a binding buffer (DMEM, 25 mM Hepes, pH 7.4, 1% BSA)containing 100 000 cpm/well of ¹²⁵I-leptin (Perkin Elmer life sciences,Paris, France) in the presence or absence of 200 nM of nonradioactiveleptin (recombinant human leptin (PeproTech Inc., USA)) for 4 h at 4° C.The cells were washed twice with PBS at the temperature of ice, lysed in1N NaOH and the radioactivity was determined in a gamma-radiationcounter. In order to measure the total amount of leptin binding in theextract, the cells were suspended in 1.5 ml of binding buffer containing0.15% of digitonin for 2 h at 4° C. The extracts were centrifuged for 30min in an Eppendorf centrifuge at maximum speed, at 4° C. Thesupernatant (0.2 ml) was incubated with 100 000 cpm of ¹²⁵I-leptin inthe presence or absence of 200 nM of leptin in a total volume of 0.25ml, with stirring overnight at 4° C.

0.5 ml of γ-globulin (1.25 mg/ml) and 0.5 ml of polyethylene glycol 6000(25% w/v) were added in order to precipitate the receptor-ligandcomplexes, which are obtained by centrifugation (17 000×g for 3 min).The pellet was washed with 1 ml of 12% (w/v) polyethylene glycol 6000,and then counted.

Activation of the Reporter Gene

HeLa cells were cotransfected with 2.6 μg of plasmids carrying the STAT3reporter gene (a gift from Dr. Levy, New York University, New York,USA), 200 pg of pcDNA3 comprising the coding region of Renillaluciferase (used as internal control) and with 1.4 μg of the various OBRconstructs or with the vehicle alone. 48 hours after transfection, thecells were depleted overnight in DMEM (1% BSA) before stimulation with 1nM of leptin for 6-8 hours. The cells were then washed and lysed in apassive lysis buffer (Promega Corporation, Madison, Wis.) for 15 minutesat ambient temperature. The total lysates were centrifuged for 2 minutesat 15 000 rpm and the supernatants were used in an assay for measuringLuciferase (Promega Corporation, Madison, Wis.) using a Bertholdluminometer (Lumat LB 9507). The results are expressed by the ratio ofthe firefly luciferase/Renilla luciferase activity.

Measurement of BRET

48 hours after transfection, HeLa, COS and HEK 293 cells expressing OBRwere detached and washed with PBS. 1-2×105 cells were distributed into96-well optical plates (Packard Instrument Company, Meriden, Conn.) inthe presence or absence of ligands, at 25° C. Membranes prepared fromcells expressing OBR were used for the measurements of BRET. Thesubstrate, coelenterazine h (Molecular Probes, Eugene, Oreg.), was addedat a final concentration of 5 μM and the reading was carried out with aFusion™ fluoro/luminometer (Packard Instrument Company, Meriden, Conn.),which permits the sequential integration of the luminescence signalsdetected with two filters (Luc filter: 485±10 nm; YFP filter: 530±12.5nm). The BRET ratio is defined as the difference in the emission at 530nm/485 nm of the cotransfected Luc and YFP fusion proteins and theemission at 530 nm/485 nm of the Luc fusion protein alone. The resultsare expressed in milliBRET units (mBU), 1 mBRET unit corresponding tothe value of the BRET ratio multiplied by 1 000. The following ligandswere used to determine the specificity of the assay: recombinant humanerythropoietin (EPO), insulin (Ins), lipopolysaccharide (LPS, SigmaAldrich, St. Louis, USA), recombinant human trombopoietin (TPO), GM-CSF,interleukin 3 (IL3), interleukin 6 (IL6), prolactin (PRL), SCF, EGF andTNFA.

EXAMPLE 1 Functional Expression of the OBR Fusion Proteins

The long form (OBR1) and the short form (OBRs) of the OBR were-fused attheir C-terminal ends with YFP or Luc (FIG. 1). The expression of thesefusion proteins was confirmed in transfected COS cells in bindingexperiments with ¹²⁵I-leptin (FIG. 2 a). Similar results were obtainedin transfected HeLa cells.

The expression, at the surface of the cells, of the fusion proteins andof wild-type receptors expressed in the COS cells vary between 5 and10%, which is in agreement with already known values. Similar values areobtained in HEK 293 cells expressing endogenous OBR (14±3%).

The location of the OBR fusion proteins in the HeLa cells was studied byfluorescence microscopy using the proteins from fusion with YFP. Thefluorescence due to OBR1-YFP is distributed in a punctate fashion in thecells whereas that due to OBRs-YFP is located in plaques. Stimulationwith leptin localized OBR1-YFP in large intracellular plaques probablycorresponding to the endosomal compartment. The location of OBRs-YFPdoes not change significantly. The results obtained by fluorescencemicroscopy confirm the predominant location of OBR in the intracellularcompartment and are coherent with the already known location of theOBR1-GFP fusion protein in COS cells.

The functional expression of the fusion proteins is evaluated bymeasuring the activation of the JAK-STAT pathway. The JAK2 kinases areassociated with intra-cellular domains of OBRs and OBR1. Ligand bindinginduces trans-phosphorylation of JAK2 and phosphorylation of OBR1, butnot of OBRS. Phosphorylated OBR1 then provides a site attachment for theSTAT proteins, which are activated by phosphorylation of the tyrosineafter binding to the receptor. The activated STAT proteins then dimerizeand are translocated to the nucleus, where they stimulate genetranscription via STAT responsive elements, as described by Tartaglia(1997, J. Biol. Chem. 272, 6093-6096).

As shown in FIG. 2 c, all the OBRs constructs induce JAK2phosphorylation, which indicates activation of JAK2. The activity of theSTAT3 reporter gene is activated 2- to 4-fold by OBR1-wt and the OBR1fusion proteins, whereas the short isoforms have no effect on theactivity of the reporter gene. These results indicate that the OBRfusion proteins are functionally expressed in the HeLa cells.

EXAMPLE 2 Constitutive Dimerization of OBR in Living Cells

The dimerization of OBR-Luc and OBR-YFP was studied in living cells.

Significant energy transfers were observed between OBRs-Luc and OBRs-YFPand also between OBR1-Luc and OBR1-YFP, expressed in equimolar amounts,which indicates that constitutive homodimers exist for the two receptors(FIG. 3 a, b). The existence of the OBRs/OBR1 hetero dimers in theliving cells is demonstrated by the detection of BRET between OBRs-Lucand OBR1-YFP, and also between OBR1-Luc and OBRs-YFP.

The specificity of these interactions is illustrated by the absence ofsignificant transfer between OBRs-Luc and OBR1-Luc and a protein fromfusion between YFP and the insulin receptor recently described (Boute etal., 2001, mentioned above).

These results indicate that the short and long isoforms are involved inhetero- and homocomplexes in living cells.

EXAMPLE 3 Effect of Leptin Binding on Constitutive BRET of the OBR

In order to evaluate the agonist effects on the constitutive BRET, thecells were preincubated with leptin before initiating the luciferasereaction with its substrate.

No change in the constitutive BRET is observed with the OBR1 homodimersand the two combinations of hetero dimers OBRs/OBR1, whereas the BRET isincreased with the OBRs homodimers (FIG. 4 a).

The changes in BRET of the OBRs homodimers induced by leptin were thenmeasured in various cell preparations. Mechanical rupture of the cellsin a hypotonic buffer significantly enhances the increase in BRET withleptin, whereas the basal BRET remains unchanged.

Similar results were obtained with the membrane fraction afterseparation from the cytosol. While all the OBRs-Luc/OBRs-YFP couplescontribute to the basal BRET, only the receptors exposed to the cellsurface (5-10%) can be stimulated by leptin, which is impermeable to themembranes in intact cells.

Disruption of the cell membranes increases the OBR fraction which isaccessible to leptin and which is responsible for the increase in theBRET induced by leptin.

Similar results were obtained on cells treated with saponin. Thiscomponent makes holes in the membranes and allows penetration ofproteins such as leptin into the intracellular compartments where themajority of the OBR are found.

No change in leptin-induced BRET was observed in similar experimentscarried out with preparations using cells expressing OBR1 homodimers—orOBRs/OBR1 hetero-dimers.

The amounts of and the ratios of OBRs-Luc and OBRS-YFP were thenmodulated in order to optimize the leptin-induced BRET (FIG. 5 a). Thebest results are obtained when 500 ng of DNA encoding OBRs-Luc and 250ng of DNA encoding OBRs-YFP are used. Under these optimized conditions,a saturated concentration of leptin induces a 2- to 2.5-fold increase inthe basal BRET signal in cells incubated with saponin or membranesprepared from cells expressing OBRs homodimers. This increase depends ontime. The maximum values are reached after 20 minutes of incubation with1 nM leptin at ambient temperature (FIG. 5 b). For higher concentrationsof leptin, the maximum values are obtained after 5 minutes of incubationat ambient temperature.

The effect of the leptin is dose-dependent, with an EC50 ofapproximately 100 pM (FIG. 5 c), which is in agreement with the Kivalues obtained with the OBRs-Luc (116 pM) and OBRS-YFP (35 pM) fusionproteins (FIG. 5 d). The specificity of the assay is demonstrated by theabsence of BRET induced by the ligand with a saturating concentration ofseveral cytokines and other membrane receptor ligands, such aserythropoietin, thrombopoietin, GM-CSF, IL3, IL6, PRL, SCFα, EGF,insulin, LPS and TNFα.

The distribution of the receptors in dimers follows statistical laws,and at a 1/1 receptor number ratio, the following distribution isexpected if all the receptors are in dimeric form: 1/4 Luc/Luc, 1/4YFP/YFP and 1/2 receptors capable of engendering a BRET signal (1/4Luc/YFP, 1/4 YFP/Luc). However, in the BRET measurements, all of themolecules fused to Luc give a luminescence signal and therefore, at a1/1 ratio, half the receptors capable of BRET are observed, on a totaldonor population. Thus, to increase the BRET signal, experiments werecarried out in which the Luc molecules were saturated with the YFPmolecules so as to have all the Luc molecules in the form of dimers withthe YFP molecules (capable of BRET). The results in FIG. 6 show that thebasal BRET signal increases during the saturation and that the inductionwith leptin is proportional to the basal signal, with a 2- to 2.5-foldstimulation of the basal BRET. At saturation, a better resolution of thebasal and induced BRET is obtained, allowing easier screening for thesearch for molecules.

1. A first fusion protein comprising a leptin receptor comprising ashort isoform comprising a Box1 intracellular domain free of a Box3intracellular domain or a soluble form of leptin receptor comprising theleptin-binding region of the molecule and an energy donor protein incombination with a second fusion protein comprising a leptin receptorcomprising a short isoform comprising a Box1 intracellular domain freeof a Box3 intracellular domain or a soluble form of leptin receptorcomprising the leptin-binding region of the molecule and an energyacceptor protein.
 2. The fusion protein combination as claimed in claim1, wherein the leptin receptor is the human OBRs isoform comprisingsequence SEQ ID No.
 2. 3. The fusion protein combination as claimed inclaim 1, wherein the leptin receptor sequence comprises amino acids 46to 866 of the human OBRs isoform of sequence SEQ ID No.
 2. 4. The fusionprotein combination as claimed in claim 1, wherein the leptin receptorsequence comprises SEQ ID No.
 4. 5. The first fusion protein as claimedin claim 1, wherein the energy donor protein is a luciferase.
 6. Thesecond fusion protein as claimed in claim 1, wherein the energy acceptorprotein is selected from the group consisting of GFP, a GFP mutant andDsRed.
 7. The second fusion protein as claimed in claim 6, wherein theGFP mutant is selected from the group consisting of YFP, EYFP, GFPS65T,or Topaz.
 8. The first fusion protein as claimed in claim 1, wherein thefusion protein sequence comprises SEQ ID No.
 6. 9. The second fusionprotein as claimed in claim 1, wherein the fusion protein sequencecomprises SEQ ID No.
 8. 10. A nucleic acid comprising SEQ ID No.
 5. 11.A nucleic acid comprising the sequence SEQ ID No.
 7. 12. An isolatedcell comprising the nucleic acid as claimed in claim
 10. 13. An isolatedcell expressing the fusion protein as claimed in claim
 1. 14. Anisolated fragment of the cell as claimed in claim 12 or 13, wherein thefragment comprises the nucleic acid of claim 12 or the fusion protein ofclaim
 13. 15. A lysate of the cell as claimed in claim 12 or 13, whereinthe lysate comprises the nucleic acid of claim 12 or the fusion proteinof claim
 13. 16. An isolated membrane of the cell as claimed in claim13, wherein the membrane comprises the fusion protein.
 17. A compositioncomprising saponin and the cell of claim 12 or 13.